To understand the relationship between hypertension and insulin resistance and the effects of nutritional supplements to improve or worsen these conditions, we are studying insulin signal transduction pathways related to nitric oxide (NO) production in vascular endothelium. Human forearm blood flow studies suggest that physiological concentrations of insulin cause vasodilation of small vessels via the production of NO in endothelial cells. Furthermore, the degree of insulin sensitivity exhibited for this response is positively correlated with insulin sensitivity for glucose uptake. Thus, insulin resistance may contribute to the pathogenesis of hypertension and other cardiovascular disorders under some conditions. We used an NO electrode to directly measure NO at nanomolar concentrations to characterize the insulin response of human umbilical vein endothelial cells (HUVEC) in primary culture. In addition, we developed a novel method for transiently transfecting endothelial cells in primary culture and selecting the transfected cells using a fluorescently activated cell sorter. Recently, we also developed a method to directly visualize NO production in single living cells using an NO-specific fluorescent dye DAF-2. By overexpressing wild-type, constitutively active, or dominant inhibitory forms of various signaling molecules, we have now elucidated a complete biochemical pathway from the insulin receptor to phosphorylation of IRS-1, leading to binding and activation of PI 3-kinase, activation of PDK-1, phosphorylation and activation of Akt that then directly phosphorylates eNOS resulting in activation of eNOS and increased NO production. Moreover, we have ruled out an important role for Ras in insulin-stimulated proudction of NO. We have also used the NO-specific fluorescent dye DAF-2 to visualize production of NO in single cells and dissect the mechanisms whereby insulin regulates activity of endothelial nitric oxide synthase. We found that insulin stimulates activation of eNOS by a calcium independent mechanism involving phosphorylation of eNOS by Akt. We have initiated studies to investigate the role of nutritional supplements DHEA and epigallocatechin gallate (EGCG, a polyphenol in green tea) to activate endothelial nitric oxide synthase and modulate or augment insulin-stimulated production of NO and ET-1. In this context, we are attempting to identify cell surface membrane receptors for DHEA and EGCG that may mediate some of these effects. In addition, we have identified novel biological actions of adiponectin and ghrelin to stimulate production of NO in endothelium and we are attempting to elucidate the respective signal transduction pathways regulating these processes.